In Yersinia pestis, the causative aaent of plague, loss of the plasmid pVW1 correlates with virtually complete loss of virulence as well as the loss of two other traits, requirement of Ca2+ or nucleotides for growth at 37 C and production of two proteins called the plague virulence antigens V and W. Because V and W re made in vitro only under conditions simulating the intracellular environment of mammalian cells, they may be necessary for intracellular survival of Y. pestis. We propose a series of genetic, physiological, and animal studies to determine if V and W antigens and Ca2+-dependence are required for virulence. We also propose to identify the genes necessary for expression of these traits, identify other genes on the pVW1 plasmid required for virulence and their products, and study the mechanisms by which these genes contribute to pathogenesis of plague. We will study virulence genes by mapping the locations of mutations in the pVW1 plasmid which cause loss of virulence and will use these mutations to detect cloned fragments of pVW1 which carry the genes. Gene products will be identified by examining the proteins produced when these fragments direct protein synthesis. Once they are identified, we will determine the location of these proteins within fractionated Y. pestis cells by electrophoretic techniques. Finally, we will characterize the properties of Y. pestis strains carrying pVW1 with virulence gene mutations, and of Y. pestis and Escherichia coli carrying virulence genes on cloned fragments. These properties will include expression of Ca2+-dependence and V and W antigens, regulation of virulence genes, interaction of the mutants with mouse macrophages in vitro, and infections caused by the mutants in mice. Our results will provide specific information about the role of pVW1 in pathogenesis of plague and will directly relate to mechanisms of pathogenesis in Yersinia enterocolitica and Yersinia pseudotuberculosis, two important human pathogens which appear to require plasmids similar to pVW1 for virulence. These studies also will provide insights of general relevance to microbial pathogenesis and mechanisms of intracellular survival.